Helmut Schubert

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: Investigation for cartilage tissue engineering

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.

Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells

Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques.

Three-dimensional visualization of in vitro cultivated chondrocytes inside porous gelatine scaffolds: A tomographic approach

Synchrotron radiation-based microcomputed tomography (SR-microCT) has become a valuable tool in the structural characterization of different types of materials, achieving volumetric details with micrometre resolution. Biomedical research dealing with porous polymeric biomaterials is one of the research fields which can benefit greatly from the use of SR-microCT. This study demonstrates that current experimental set-ups at synchrotron beamlines achieve a sufficiently high resolution in order to visualize the positions of individual cartilage cells cultivated on porous gelatine scaffolds made by a freeze-structuring technique. Depending on the processing parameters, the pore morphology of the scaffolds investigated was changed from large-pore sized but non-ordered structures to highly directional and fine pored. The cell-seeded scaffolds were stained with a combined Au/Ag stain to enhance the absorption contrast in SR-microCT. While only some cells showed enhanced absorption contrast, most cells did not show any difference in contrast to the surrounding scaffold and were consequently not detectable using conventional greyscale threshold methods. Therefore, using an image-based three-dimensional segmentation tool on the tomographic data revealed a multitude of non-stained cells. In addition, the SR-microCT data were compared with data obtained from scanning electron microscopy, energy dispersive X-ray spectroscopy and histology, while further linking the initial cell density measured via a MTT assay to the pore size as determined by SR-microCT.

Immobilization and controlled release of prostaglandin E2 from poly-L-lactide-co-glycolide microspheres

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite involved in physiological homeostasis and numerous pathophysiological conditions. Furthermore, it has been demonstrated that prostaglandins have a stimulating effect not only on angiogenesis in situ and in vitro but also on chondrocyte proliferation in vitro. Thus, PGE(2) represents an interesting signaling molecule for various tissue engineering strategies. However, under physiological conditions, PGE(2) has a half-life time of only 10 min, which limits its use in biomedical applications. In the present study, we investigated if the incorporation of PGE(2) into biodegradable poly-L-lactide-co-glycolide microspheres results in a prolonged release of this molecule in its active form. PGE(2)-modified microspheres were produced by a cosolvent emulsification method using CHCl(3) and HFIP as organic solvents and PVA as emulsifier. Thirteen identical batches were produced; and to each batch 1.0 mL of serum-free medium was added. The medium was removed at defined time points and then analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) to measure the residual PGE(2) content. In this study we demonstrated the prolonged release of PGE(2), showing a linear increase over the first 12 h, followed by a plateau and a slow decrease. The microspheres were further characterized by scanning electron microscopy.

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning.

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.

Characterization of oriented protein-ceramic and protein-polymer-composites for cartilage tissue engineering using synchrotron μ-CT

Abstract In this paper we report on the synthesis of three different gelatine based scaffold materials for the reconstruction of articular cartilage defects. The first scaffold design is based on an unmodified, oriented gelatine network, while the second design further comprises an attached inorganic hydroxyapatite layer and the third design includes poly(l-lactide) microspheres as a model material for future drug-release applications. All three scaffold designs were characterized and imaged using synchrotron μ-CT, obtaining a complete volumetric reconstruction of a previously defined sample region. Furthermore, two unmodified scaffolds were cultivated for one week with porcine chondrocytes. Afterwards the attached cells were labelled using a combination of Au-lysine and silver enhancer. In synchrotron μ-CT analysis we were thus able to map the cell distribution due to the difference in X-ray absorption of the labelled cells and the non labelled scaffolds in a volume of several millimetres.

Perspectives for the tissue engineering of cartilage from a biological and biomaterial point of view.

Tissue engineering has become a fast growing interdisciplinary branch of research at the interface between life and engineering sciences with important clinical end-points. In this context the regeneration of articular cartilage represents an exciting challenge since hyaline cartilage has a limited capacity for self-repair. Today the use of different scaffold materials combined with in vitro expanded chondrocytes and signalling molecules poses great hopes for an optimal treatment of articular cartilage defects. However, until today the optimal construct of scaffolds, cells and signalling molecules has not yet been found. Since repair and regeneration recapitulate in part ontogenetic processes, the present paper summarizes the regulative mechanisms of endochondral ossification in the growth plate of the long bones to identify possible new signalling molecules for the improvement of tissue engineering-based solutions in the treatment of cartilage defects. The growth plate represents a highly organized structure of chondrocytes and extracellular matrix components in distinguishable proliferation and differentiation stages. It is regulated by various paracrine and hormonal factors. In a second part we present actual trends in scaffold design based on synthetic polymers and natural polymers, stressing their potential use in the regeneration of cartilage defects from the point of view of bioactivity and biocompatibility. In conclusion, both new signalling molecules from basic research and innovative scaffold materials with variable physico-chemical properties open up new and interesting perspectives for the research in optimized tissue engineeredbased therapeutic strategies to treat cartilage defects.

Oriented Collagen-Based/Hydroxyapatite Matrices for Articular Cartilage Replacement

Mechanical stimulation of articular cartilage is known to be es sential for chondrogenesis. In our study we use animal cartilage-bone explants from the femur condylus, which were divided into three experimental groups (loaded, unloaded and controls). Loading was pe rformed in a sterile and physiological environment based on DME-medium. Cell morphology and cell vitality was observed by histological techniques. The knowledge gathered from the mecha nical in-vitro study is now used to produce a structured artificial matrix resembling nati ve cartilage in biochemical composition, biomechanical properties and zonal tissue structure. The art ificial matrix is then seeded with chondrocytes and loaded with the same parameters we found t o be ideal for tissue regeneration. Analysis is performed using histological and immuno-histological m ethods.

A method to screen and evaluate tissue adhesives for joint repair applications

BackgroundTissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications.MethodTissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives.ResultsViability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa).ConclusionThe method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.

Synchrotron µCT Investigation of the Collapsing Pore-Network of Gelatin Scaffolds under Compression

Biomaterials based tissue engineering requires optimization of several parameters. The most important parameters can be attributed to biocompatibility, degradational behaviour, mechanical stability and structural design. In previous studies we have established a porous gelatine based scaffold material, with parallel oriented pore channels. Although, tomographic data has been derived on dried scaffold samples, it remains unclear how the pore channel network interacts under load in a wet environment. We developed an experimental setup to compress biomaterials in a wet environment during exposure to synchrotron generated X-rays using a micrometer screw with a force sensor. Achieving good X-ray absorption contrast in polymeric biomaterials immersed in water is rather difficult, as water absorption prevents detailed imaging. Phase contrast imaging on the other hand allows for improved imaging results due to the attenuation of phase boundaries in the imaged data, neglecting effects of X-ray absorption in the watery phase nearly completely. Best results were obtained for X-ray energies of 30 keV with a scintillator to sample distance of 1090 mm with the established experimental setup. Due to over attenuation of phase boundaries at higher beam energies, this energy was finally chosen. As a result, we could evaluate the collapsing pore network upon loading with the possibility to enhance the structural design for future studies.